Assay and method for quantifying the levels of steryl esters and free sterols in starch-containing food products

ABSTRACT

The present invention relates to an assay for determining the levels of sterols, stanols, steryl esters, fatty acid derivatives and combinations thereof in a starch-containing food product. The assay is particularly useful in supporting product health and/or nutritional claims in manufacturing products intended for human or animal consumption. The present invention describes a method for extracting sterols related compounds and uses as an internal standard a steryl ester, preferably cholesterol oleate. By using the present extraction technique the process enables the recovery of substantially all of the sterol related compound in the sample.

CROSS-REFERENCES TO RELATED APPLICATIONS

None.

BACKGROUND OF THE INVENTION

Manufacturers of consumer goods, specifically, manufacturers of foodproducts annually devote a substantial amount of time, effort andresources to improving the products they offer. These improvements cantake the form of improved taste or texture, reduced calories or fatcontent and the like. More recently, and as the population matures, foodcompanies are looking more and more into food products, food componentsor ingredients that deliver a particular health or nutritional benefit.That is, food products that may assist the consumer in living ahealthier life in that the food product aids in reducing highcholesterol levels, mitigating the risk of heart diseases, diminishingthe risk of some cancers and many other illnesses and diseases, whichbecome more prevalent as society ages or dietary patterns are modifiedto meet the changing lifestyles of today's population.

One health claim that has received a lot of interest lately is theeffect of using sterols/stanol, steryl esters and other fatty acidderivatives and combinations thereof to reduce unhealthy or highcholesterol levels. In this regard, a number of internationally knownfood manufacturers have successfully manufactured and marketedstarch-containing food products that have certain levels of sterols andsteryl esters in order to deliver a product that provides this healthbenefit. Such starch-containing food products include ready to eat (RTE)cereals, dough based products, RTE meals and the like.

It is well known that cholesterol in humans comes from primarily twosources, the body's own production of cholesterol (endogenous) anddietary cholesterol (exogenous). Typically, the average person consumesbetween 350-400 milligrams of cholesterol daily, while the recommendedintake is around 300 milligrams. Increased dietary cholesterolconsumption, especially in conjunction with a diet high in saturated fatintake, can result in elevated serum cholesterol. Elevated serumcholesterol is a well-established risk factor for heart disease andtherefore there is a need to mitigate the undesired effects ofcholesterol accumulation. High cholesterol levels are generallyconsidered to be those total cholesterol levels at 200 milligrams perdeciliter and above or LDL cholesterol levels at 130 milligrams perdeciliter and above.

Lipoproteins contain specific proteins and varying amounts ofcholesterol, triglycerides and phospholipids. There are three majorclasses of lipoproteins and they include very low density lipoproteins(“VLDL”), low density lipoproteins (“LDL”) and high density lipoproteins(“HDL”). The LDLs are believed to carry about 60-70% of the serumcholesterol present in an average adult. The HDLs carry around 20-30% ofserum cholesterol with the VLDL having around 1-10% of the cholesterolin the serum. To calculate the level of non-HDL cholesterol present(find the level of LDL or VLDL levels), which indicates risk, the HDL issubtracted from the total cholesterol value. By lowering the totalsystem LDL cholesterol level, it is believed that certain health risks,such as coronary disease and possibly some cancers, that are typicallyassociated with high cholesterol levels, can be reduced.

Numerous studies relating to modifying the intestinal metabolism oflipids have been done to illustrate that such effects can reduce a highcholesterol level. This may be done by hampering the absorption oftriglycerides, cholesterol or bile acids. It is believed that certainplant sterols, steryl esters, stanols fatty acid derivatives andcombinations thereof lower serum cholesterol levels by reducing theabsorption of dietary cholesterol and/or bile acids from the intestines.

Sterols occur in natural fats and oils, particularly in vegetable oils.Unsaturated vegetable oils and non-animal fat oils, such as soybean oil,wheat germ oil, cottonseed oil, safflower oil, peanut oil, rice oil,canola oil and the like are well known sources of β-sitosterol,stigmasterol, ergosterol and campesterol as well as various othermaterials such as higher aliphatic alcohols. Tall oil is also asignificant source of β-sitosterol and campesterol.

Stanols (β-sitostanol, campestanol, stigmastanol and fatty acidderivatives thereof) are the 5 alpha saturated derivatives of plantsterols and may be derived from similar sources set forth above.

Natural plant sterols are similar structurally to cholesterol except inthe arrangement of the basic side chains. Absorption of plant sterols inthe intestines is believed to be minimal at best and sterols/steroidsare generally excreted in the stool. Thus, the levels of plant sterolsin the serum are relatively low since they are not absorbed by the bodyand are relatively quickly excreted. Where the amount of sterols isincreased in an effort to obtain greater beneficial or health effects,the sterols still do not increase significantly in amount in the bloodserum as the absorption capability, however limited it may be, isquickly exceeded. Hence, the interest in including sterol related orcontaining compounds in food products, food ingredients and foodcomponents (the presumed health benefit stated above) is directlyrelated to the manufacturer's interest in sterol inclusion into suchproducts.

In manufacturing products that contain certain health claims, such as aRTE cereal, i.e. TOTAL® or CHEERIOS® available from General Mills, Inc.of Minneapolis, Minn., it is important that the product not only be ableto support or substantiate the health claim for regulatory reasons butalso that the product must actually contain the amount of the effectiveingredient stated in the nutritional information provided with thepackage. One of the problems associated with making foods having sterolrelated or containing compounds is determining or verifying the actuallevel of sterol related or containing compounds in the end product to beconsumed.

Thus, there is a need for food manufacturers to be able to accuratelycalculate or quantify the amount of the sterols and steryl esters in afood product so that the proper amount is delivered in each of thesuggested serving or portion sizes in order that the claims of the foodproduct are supported by the contents.

Heretofore, a number of methods using a variety of internal standardshave been developed to attempt to calculate the amount of sterol relatedor containing compounds in the food product. However, these methodswhile possibly being relatively quick and inexpensive to use can in factbe detrimental to both the manufacturer from both a cost and aregulatory standpoint as well as the consumer of the product from ahealth related aspect.

With respect to prior methodologies employed by the food productmanufacturer, readings related to determining the level of sterol basedor containing compounds provided by these methods often resulted in areading that was substantially lower than the actual amount of sterolsor steryl esters that may have been added to the food products duringthe manufacturing process, that is, that amount added to provide orobtain the health or nutritional benefit. Often, it was found thatreadouts from these prior test methods would be from twenty-five tofifty percent (25-50%) lower than the actual amount of the ingredient orcomponent that was added. Such readings would then result in themanufacturer adding even more of the ingredient or component to insurethat the food product would be supported by the claims and nutritionalinformation provided with the package, that is in the present example,to insure that enough of the sterol based compound is present. Whilethis is a simple solution, it has significant economic disadvantages inthat sterol related compounds are relatively expensive ingredients whencompared on a relative weight basis with other ingredients present inthe product, i.e. the grain (wheat, oat, barley), sugar, macro and micronutrients, etc. There are also other significant disadvantages to havingexcessive amounts of sterol containing compounds in the food products.As used herein, the term “food products” includes food components (suchas dough, flakes), food intermediates (a transitional step used inmaking a product or component) and a food ingredient. Where the term“food product” is used in connection with a manufacturer ormanufacturing, the term is intended to imply an entity, which makes,fabricates, processes or produces food products as defined above.

It is important to insure that the right amount of the ingredient isavailable in each serving or portion size, not only from a manufacturingbut also a regulatory standpoint. From a manufacturing point of view,cost of manufacture is a significant factor in determining profitlevels. However, from a regulatory standpoint, providing an amount ofsterols and steryl esters beyond an acceptable threshold can bedetrimental to the health of the consumer. As such, there is a maximumamount of the ingredient that cannot be exceeded in order to qualify forthe generally recognized as safe (“GRAS”) status of such sterol relatedcompounds. As used herein, the term sterol related or containingcompound refers to such compounds as β-sitosterol, stigmasterol,ergosterol, campesterol, stanols β-sitostanol, campestanol, stigmastanoland fatty acid derivatives thereof.

It is believed that the prior testing methods, which used an internalstandard of cholestane, produced unacceptable results in part due to thefact that cholestane did not sufficiently chemically simulate thecompounds that were being assayed to compensate for degradation ormatrix binding. The problem of getting an accurate reading using currentmethods is that the steryl esters and sterols are bound by the starch(most probably amylopectin) during the cooking process, which isinherent in most food product manufacturing steps. Using such internalstandards prior test methods were ineffectual in releasing the starchbound sterols and steryl esters resulting in readings being off by asmuch as fifty percent (50%) from the known amount of ingredient orcomponent that was added to the food product or food componentinitially.

What is needed therefore is an accurate method for determining orquantifying the level of the sterol related or containing compounds in aparticular starch containing or consumer food product or component whichhas a health claim or benefit associated with it so as to insurecompliance with self-affirmed GRAS status as well as to maintainmanufacturing economies in scale with target projections.

SUMMARY OF THE PRESENT INVENTION

The embodiments of the present invention described below are notintended to be exhaustive or to limit the invention to the precise formsdisclosed in the following detailed description. Rather, the embodimentsare chosen and described so that others skilled in the art mayappreciate and understand the principles and practices of the presentinvention.

In a presently preferred embodiment of the present invention, theinvention comprises an extraction method to be able to quantify theamount of sterol based compounds in starch-containing food product,which includes the steps of providing an amount of a steryl ester,preferably cholesterol oleate, and then initially hydrating apolysaccharide matrix of a starch-containing food product. Then mixingthe matrix with an organic solvent. The organic phase is separated froman aqueous phased which was created by the mixing process. The organicphase is then subject to a drying step in order that the level or amountof sterol based compounds in the starch containing food product can bedetermined.

A still further preferred embodiment of the present invention sets forthan assay for determining quantities of sterols, stanols, steryl esters,fatty acid derivatives and combinations thereof in a cereal basedproduct using a steryl ester as an internal standard. Preferably, thesteryl ester is cholesteryl oleate.

A additional preferred embodiment of the present invention relates to anassay for determining quantities of sterols, stanols, steryl esters,fatty acid derivatives and combinations thereof in which more than 90%,preferably closer to 100%, of the sterols, stanols, steryl esters, fattyacid derivatives and combinations thereof are recovered forquantification.

Another embodiment of the present invention includes a method forsubstantiating the presence or absence of sterol based compositions thatare found in a consumer food product. This method comprises the stepsof, initially obtaining a consumer food product and then separating theconsumer food product into certain fractions. Once the fractions areseparated, at least one of the fractions are hydrated. Next, an organicsolvent is added to the hydrated fraction. This hydration creates anorganic phase which is sequestered from the organic phase of thehydrated fraction. Using an internal standard of a sterol relatedcompound the amount of sterol based compounds in the consumer foodproduct is determined. Once the amount of sterol based compound isdetermined that result is communicated to deliver a particular healthmessage related to the presence of the sterol based compound.

A still further embodiment of the present invention relates to a methodof reporting a health benefit of a consumer food product. This methodcomprises the steps of initially manufacturing a food product that isintended for human consumption. (Although only human consumption isreferenced herein, this embodiment as well as the other embodimentsprovided in this application may include food products that are intendedfor animal consumption as well.) In order to determine the presence orabsence of a sterol related compound in the food product, an internalstandard of a sterol related compound is provided as a reference. Thefood product is then hydrated to create a matrix through the addition ofan organic solvent. An organic phase of the matrix is separated andanalyzed to obtain a level of sterol related compounds found in thehydrated food product. Finally, once the amount of sterol relatedcompounds in the food product is known, a health benefit of the foodproduct containing sterol related compounds is advertised to potentialconsumers of the health product.

Publications, patents and patent applications are referred to throughoutthis disclosure. All references cited herein are hereby incorporated byreference. All percentages and ratios are calculated by weight unlessotherwise indicated. All percentages and ratios are calculated based onthe total composition unless otherwise stated.

These, as well as other objects and advantages of this invention, willbe more completely understood and appreciated by referring to thefollowing more detailed description of the presently preferred exemplaryembodiments of the invention in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

These, as well as other objects and advantages of this invention, willbe more completely understood and appreciated by referring to thefollowing more detailed description of the presently preferred exemplaryembodiments of the invention in conjunction with the accompanyingdrawings, of which:

FIG. 1 is a gas chromatograph illustrating sterol standards; and

FIG. 2 is a graph showing the correlation of the level of steroldetermined by the prior method to the amylopectin content of cookeddoughs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is now illustrated in greater detail by way of thefollowing examples, but it should be understood that the presentinvention is not to be construed as being limited thereto.

In the preferred embodiment, a starch containing food product, such asan RTE cereal is prepared in a conventional manner. This exemplary RTEcereal is in the form of flakes that are created by preparing a cookedcereal dough through known methods and then forming the cooked cerealdough into pellets that have a desired moisture content. The pellets arethen formed into wet flakes by passing the pellets through chilledroller and then subsequently toasting or heating the wet cereal flakes.The toasting causes a final drying of the wet flakes, resulting inslightly expanded and crisp RTE cereal flakes. The flakes are thenscreened for size uniformity. The final flake cereal attributes ofappearance, flavor, texture, inter alia, are all affected by theselection and practice of the steps employed in their methods ofpreparation. For example, to provide flake cereals having a desiredappearance feature of grain bits appearing on the flakes, one approachis to topically apply the grain bits onto the surface of the flake aspart of a coating that is applied after toasting.

In the following example, the assay is based on using an internalstandard of cholestryl oleate instead of cholestane, which has beenperceived as producing unacceptable results as typified in the graphillustrated in FIG. 2. As set forth previously, it is believed thatcholestane did not sufficiently, chemically simulate the compounds thatwere being assayed to compensate for degradation or matrix binding whichfurther exacerbates the problem of getting an accurate reading in thatthe steryl esters, stanols and sterols are bound by the starch (mostprobably amylopectin) during the cooking process. By using the followingprocess substantially all, that is greater than 90%, but typicallycloser to 100% of the sterol related compounds that is added to thesample product is recovered.

Example A Extraction of Steryl Esters/Sterols/Stanos from Cooked CerealProducts:

-   1. Add Cholesterol Oleate (0.1 mg) to a clean 20 ml glass    scintillation vial with phenolic foil lined cap (“Vial”).-   2. Weigh out 0.1000 gram of sample into the Vial.-   3. Add 2.0 ml of Solution 0.1 M Acetic Acid with 0.7% (w/v) KCl to    hydrate the polysaccharide matrix of the sample.-   4. Heat Sample at 95° C. for 30 minutes.-   5. Cool sample to room temperature.-   6. Add 10 ml of HPLC grade chloroform to the sample. Alternatively,    add 10 ml of 2:1 Chloroform:MeOH. Seal cap tightly. Incubate at    37° C. for 12-16 hours with constant shaking at 250 rpm.-   7. Focus organic and aqueous phase by centrifuging for 10 min at    5000 rpm separating the organic phase from the aqueous phase.-   8. Withdraw bottom layer (chloroform or chloroform:MeOH) from the    vial with a 10 ml gas tight syringe. Avoiding the aqueous layer    during the draw. Transfer to a clean flask and dry down.-   9. Transesterify using Alltech MethPrep II (Alltech Associates,    Inc., Deerfield, Ill. 60015, USA) or sodium methoxide.    Alternatively, saponify samples.-   10. Silanate samples and inject on to GC with FID detector (Hewlett    Packard 5890 Gas Chromatograph; Agilent Technologies Palo Alto,    Calif. 94303 USA ) or Mass Spec detector(Hewlett Packard Model 5970    MSD; Agilent Technologies Palo Alto, Calif. 94303 USA) to quantify    or determine the amount or level of sterol related compounds that    are found in sample.

The following example uses enzymes to digest the carbohydrate andprotein matrix and is thought to obtain substantially all, that isgreater than 90%, but typically closer to 100% of thesterol/stanol/steryl ester that is added to the sample product isrecovered.

Example B Extraction of Steryl Esters/sterols/stanols from Cooked CerealProducts

-   1. Add Cholesteryl Oleate (0.1 mg) to a clean 20 ml glass    scintillation vial with phenolic foil lined cap (Vial).-   2. Weigh out 0.1000 gram of sample into Vial.-   3. Add 2.0 ml of Solution A-2 to hydrate the polysaccharide matrix    of the sample (see below).-   4. Heat Sample at 95° C. for 30 minutes.-   5. Cool Sample to 50° C. then add 400 ul of Enzyme Solution 1 (see    below). Incubate at 50° C. for 3 hours. Vortex occasionally.-   6. Add 100 ul of Enzyme Solution 2 (see below). Incubate at 50° C.    for 1 hour. Vortex occasionally.-   7. Add 20 ul of 50% Acetic acid (Final Conc. ˜0.1 M Acetic Acid).-   8. Add 10 ml of HPLC grade chloroform. Seal cap tightly. Incubate at    37° C. for 12-16 hours with constant shaking at 250 rpm.-   9. Focus organic and aqueous phase by centrifuging for 10 min at    5000 rpm separating the organic phase from the solution.-   10. Withdraw bottom layer (chloroform) from the vial with a 10 ml    gas tight syringe. Be sure not to draw any of the aqueous layer into    the syringe. Transfer to a clean flask and dry down.-   11. Transesterify using Alltech MethPrep II (Alltech Associates,    Inc., Deerfield, Ill. 60015, USA) or sodium methoxide.    Alternatively, saponify samples to determine the amount or level of    sterol related compounds in the food product sample.-   12. Silanate samples and inject on to GC with FID(Hewlett Packard    5890 Gas Chromatograph; Agilent Technologies Palo Alto, Calif. 94303    USA) detector or Mass Spec detector.    Solution A2:    -   20 mM KH₂PO₄, pH 7.2    -   150 mM NaCl    -   50 mM KCl        Enzyme Solution 1: (make just prior to use)    -   2 grams α-Amylase    -   4 ml Amyloglucosidase (volume to 500 ml with Solution A2—above)        Enyme Solution 2: (make just prior to use)    -   1 gram Papain    -   0.6 grams Dithiothreitol (19.5 mM)    -   volume to 200 ml with Solution A2 (above)

In order to determine the amount of sterols, stanols, steryl esters,fatty acid derivatives or combinations thereof which have been recoveredby using one of the aforementioned examples, the sample is subjected togas chromatography. FIG. 1, illustrates the standard peaks for sterols.

Cooked starch-containing dough samples with increasing concentrations ofamylopectin and fixed amounts of added steryl esters were assayed usingone of the above referenced processes but having cholestane as theinternal standard. FIG. 2, illustrates that the assay using the internalstandard of cholestane underestimates the amount of steryl ester in awell correlated manner (r²=0.993) to the total amount of amylopectinpresent in the sample.

The following table lists the results of exemplary RTE cereal samples,which were tested after using the process described in the abovereferenced examples. Example A lists values obtained using the processcontained herein whereas Example B lists values obtained usingcholestane as an internal standard over which the method of the presentinvention is an improvement over.

TABLE 1 Sterol Determinations Sample Name Sterol Target Example AExample B Batch Flake 2.00 2.05 ± 0.16 1.49 ± 0.10 Clinical James Flake4.10 3.93 ± 0.2  2.87 ± 0.07 James Flake 1715 3.00 3.04 ± 0.12 1.75 ±0.23 James Flake 1915 3.20 3.14 ± 0.05 1.92 ± 0.14 HSE Flake 11001 2.502.53 ± 0.01 1.56 ± 0.09 LSE Flake 12501 2.00 2.09 ± 0.05 0.87 ± 0.18

In conducting a comparison of standard steryl esters processed in theabsence of a cereal matrix by the process set forth above using eitheran internal standard of cholesteryl oleate or cholestane the followingresults were reported in Table 2. These results demonstrate that thecholestane is being recovered in higher yield relative to the silatedsterol standards resulting in a lower value for the measured sterolsthat use the cholestane internal standard. Unlike cholesteryl oleate,cholestane is unesterified during the initial extraction steps and willtherefore have different affinity for compounds and/or matrices that mayirreversibily bind steryl esters. Following the saponification ortransesterification, cholesteryl oleate and the other steryl esters willyield a free sterol with a hydroxyl group at the 3 position of thesterol ring. This hydroxyl group can cause the free sterol toirreversibly bind to glassware being used in the assay. Cholestane lacksthe hydroxyl at the 3 position. This may also explain its higher yieldrelative to the measured sterols.

TABLE 2 Sterol Determinations Sample Name Sterol Target Example B SterolStds (cholestane) 100.00 89.96 ± 0.64 Sterol Stds (cholesteryl oleate)100.00 98.41 ± 1.58

In table 3, the first column represents the sample being tested. In thistable, in addition to RTE cereals (batch flake and clinical test), adough, which may be used for breads, muffins and other baked goods isalso tested. The second column represent the sterol related compoundbased target, the third column using the process described herein andthe fourth column represents the percent difference of second and thirdcolumns.

TABLE 3 Sterol Determinations Sample Name Sterol Target Test A %Difference Brabender Var 1 2.32 2.38 ± 0.03 2.6 Brabender Var 2 3.083.08 ± 0.14 2.0 Brabender Var 3 3.71 3.98 ± 0.21 7.2 Barbender Var 44.46 4.50 ± 0.06 0.8 James Flake 2.94 3.09 ± 0.16 5.1

It is believed that each of the foregoing tables illustrate thesignificant improvement of recovery of sterol related compounds fromstarch-containing food products or food components, when compared withprevious methods that have used cholestane as the internal standard havenot hydrated the starch matrix prior to extraction.

As can be seen from the tables, by using the novel assay of the presentinvention significantly more of the sterol related compounds arerecovered when compared with previous or prior art methods.

In practicing the method embodiments of the present invention applicantsthe resultant reports are then provided in a manner that enables thecommunication or advertising of the benefit of having various levels oramounts of sterol related compounds in the food products. Thisadvertising or communication can take any number of forms including theprinting of the benefit of sterol compounds for the reduction ofcholesterol levels on the packaging of the food product, through the useof audiovisual devices such as television, computer enabled devices andprinted indicia such as newspapers, magazines, newsletters and the like.

The methods of the present invention as well as the assay itself isuseful in the determination of the levels of sterol related compounds inready to eat food products. Ready to eat food products (RTE) for thepurposes of this invention include baked goods, salted snacks, specialtysnacks and confectionary snacks as well as dairy products. Many of theaforementioned products can be dough based products, that is a dough iscreated, usually from a mixture of flour, water and other ingredients tonecessary for the finished product.

It will thus be seen according to the present invention a highlyadvantageous test methodology has been provided. While the invention hasbeen described in connection with what is presently considered to be themost practical and preferred embodiment, it will be apparent to those ofordinary skill in the art that the invention is not to be limited to thedisclosed embodiment, that many modifications and equivalentarrangements may be made thereof within the scope of the invention,which scope is to be accorded the broadest interpretation of theappended claims so as to encompass all equivalent processes,methodologies and assays.

1. An assay for determining quantities of sterol related compounds in astarch-containing food product comprising: providing an amount of asteryl ester as an internal standard; adding said internal standard to afood product containing a polysaccharide matrix; hydrating a saidpolysaccharide matrix of said food product; mixing said hydratedpolysaccharide matrix with an organic solvent; de-esterifying the sterylesters in the organic solvent; quantifying at least a level of eithersteryl esters and free sterols in said food product by a method usingsaid steryl ester as an internal standard.
 2. The assay of claim 1,wherein the organic solvent is chloroform.
 3. The assay of claim 1,wherein the organic solvent is a mixture of chloroform and methanol. 4.The assay of claim 3, wherein the mixture of chloroform and methanol isprovided in a ratio of 2 to
 1. 5. The assay of claim 1, wherein thesteryl ester is cholesteryl oleate.
 6. The assay for determiningquantities of sterol related compounds in a starch-containing foodproduct as recited in claim 1, wherein the steryl ester is cholesteryloleate.
 7. The assay as recited in claim 1 wherein the assay is used todetermine levels of sterols, stanols, steryl esters, fatty acidderivatives and combinations thereof in a cereal based product.
 8. Theassay as recited in claim 1 wherein the starch-containing food productis a ready to eat cereal.
 9. The assay as recited in claim 1 wherein thestarch-containing food product is a dough.
 10. The assay as recited inclaim 1 wherein at least 90% of the sterols, stanols, steryl esters,fatty acid derivatives and combinations thereof is recovered forquantification.
 11. The assay of claim 1 further comprisingcommunicating the amount of sterol based compounds found in saidconsumer based food products.
 12. The assay of claim 1, wherein the stepof de-esterifying the steryl esters comprises separating the organicphase from an aqueous phased created by said mixing of said matrix withsaid organic solvent; and drying said organic phase.